Methods and compositions for therapeutic treatment of cathespin k complex-mediated disorders

ABSTRACT

The present invention relates to methods and compositions for the amelioration of symptoms mediated by the collagenolytic activity of cathepsin K complex. The invention provides methods of specifically modulating the collagenolytic activity of cathepsin K without substantial interference in other biologically-relevant activities of cathepsin K.

STATEMENT OF RELATED APPLICATIONS

[0001] This application claims priority under 35 USC § 119(e) to USSN60/324,445 filed Sep. 24, 2001, the contents of which application areherein specifically incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to therapeutic methods, screeningmethods, and composition comprising a cathepsin K complex exhibitingspecific collagenolytic activity.

[0004] 2. Related Art

[0005] Cathepsin K is a cysteine protease of the papain family that ishighly expressed in bone and cartilage-degrading cells such asosteoclasts, giant multinucleated cells and synovial fibroblasts. It isestimated that cathepsin K constitutes approximately 98% of all cysteineprotease activities expressed in osteoclasts, and thus may be primarilyresponsible for most of the proteolytic activity in bone resorption.

[0006] Cathepsin K has a unique collagenolytic activity that allows theefficient degradation of native collagen fibers. Since type I collagenconstitutes more than 90% of the organic bone matrix and type IIcollagen is the major protein in cartilage, collagen degradation isthought to be the major biologically-relevant activity of cathepsin K.

[0007] Cathepsin K has been identified as a novel pharmaceutical targetfor the design of anti-resorptive drugs. The main indications for theuse of cathepsin inhibitors are osteoporosis, Paget's disease,rheumatoid arthritis, and possibly osteoarthritis. The common feature ofthese diseases is the excessive and irreversible degradation ofextracellular bone and cartilage matrix.

[0008] Cathepsin K is expressed in a variety of additional cell typessuch as macrophages, and various epithelial cells with potentialfunctions in antigen processing, thyroglobulin processing and otherfunctions yet to be determined. Thus, active-site directed inhibitorsagainst cathepsin K which result in inhibition of excessiveextracellular matrix degradation, will also inhibit many otherbiologically important cathepsin K activities. Thus, inhibition ordisruption of non-deleterious cathepsin K activities may result inundesirable and deleterious effects.

[0009] It would be useful to specifically inhibit a single cathepsin Kactivity, for example, collagen degradation, without inhibiting otherbiologically-relevant activity.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention is based, in part, on the discovery thatthe collagenolytic activity of cathepsin K requires the formation of anoligomeric cathepsin K complex from monomeric (free) cathepsin K andspecific glycosaminoglycans. Thus, the high specificity of cathepsin Kfor triple-helical collagen is a property of the complexed proteinformed from specific component glycosaminoglycans, and is not exhibitedby the free protein or a complex formed from non-productiveglycosaminoglycans. Accordingly, the present invention provides methodsfor specifically modulating the collagenolytic activity of cathepsin Kwithout inhibiting other biologically-relevant activities of cathepsinK.

[0011] In a first aspect, the invention provides a substantially purecollagenolytically active cathepsin K oligomeric complex that degradestype I and type II collagen with a high specificity relative to that offree cathepsin K, which has no collagenolytic activity. The cathepsin Kcomplex of the invention may comprise the native mature cathepsin Kpeptide, or other cathepsin K molecules, such as analogs, variants, andfragments thereof, complexed with one or more specificglycosaminoglycans, such that the complex exhibits increasedcollagenolytic activity over monomeric (free) cathepsin K.

[0012] In one embodiment, a collagenolytically active cathepsin Kcomplex of the invention comprises 2-10 cathepsin K peptides and 2-10glycosaminoglycan molecules. In a more specific embodiment, acollagenolytically active cathepsin K complex of the invention comprises5 cathepsin K peptides and 5 glycosaminoglycan molecules. In a morespecific embodiment, the glycosaminoglycan molecule is chondroitinsulfate (CS).

[0013] The cathepsin K complex of the invention can also be used toproduce antibodies that are immunoreactive or bind epitopes of thecollagenolytically active cathepsin K complex. Accordingly, in a secondaspect, the invention features antibodies to a cathepsin K complex ofthe invention. The antibodies of the invention include polyclonalantibodies which consist of pooled monoclonal antibodies with differentepitopic specificities, as well as distinct monoclonal antibodypreparations. Monoclonal antibodies are made from antigen-containingfragments of the cathepsin K polypeptide by methods known in the art(see, for example, Kohler et al. (1975) Nature 256:495).

[0014] The cathepsin K complexes of the invention are useful to screenreagents capable of specifically modulating the collagenolytic activityof a cathepsin K complex without effecting other biological activitiesof the free peptide. Accordingly, in a third aspect, the inventionfeatures methods for identifying a reagent which modulatescollagenolytically active cathepsin K complex activity, by incubating acathepsin K complex with the test reagent and measuring the effect ofthe test reagent on cathepsin K complex activity. Specific modulationembodiments include upregulation, e.g., increasing or enhancing thecollagenolytic activity of a cathepsin K complex, and downregulation,e.g., inhibiting or decreasing the collagenolytic activity of acathepsin K complex.

[0015] The cathepsin K complexes of the invention are also useful toscreen reagents capable of modulating the formation of acollagenolytically active cathepsin K complex. Accordingly, in a fourthaspect, the invention features methods for identifying a reagent whichmodulates collagenolytically active cathepsin K complex formation, byincubating free complex components, e.g., free cathepsin K andglycosaminoglycans capable of forming a collagenolytically active(productive) complex with cathepsin K, with the test reagent andmeasuring the effect of the test reagent on cathepsin K complexformation. Reagents thus identified may modulate cathepsin K complexformation by increasing or enhancing complex formation, or inhibiting,decreasing, or blocling formation of a collagenolytically activecomplex. Reagents may also modulate cathepsin K complex formation byfavoring formation of a non-productive complex (i.e.,non-collagenolytic)over formation of a productive complex.

[0016] In a fifth aspect, the invention further features a method oftreating a cathepsin K complex-mediated disorder by administering to asubject in need thereof an effective dose of a therapeutic reagent thatalters or effects cathepsin K complex activity. In one embodiment, adisorder results from excessive cathepsin K complex activity and thetherapeutic agent specifically inhibits the collagenolytic activity of acathepsin K complex. In another embodiment, a disorder results fromexcessive cathepsin K complex activity and the therapeutic agentspecifically inhibits formation of a collagenolytically active(productive) cathepsin K complex.

[0017] In one embodiment, a disorder results from insufficient cathepsinK complex activity, and the therapeutic agent specifically enhances thecollagenolytic activity of a cathepsin K complex. In a relatedembodiment, a disorder results from insufficient cathepsin K complexactivity and the therapeutic agent specifically increases formation of acollagenolytically active cathepsin K complex.

[0018] The materials of the invention are ideally suited for thepreparation of a kit for the detection of the level or activity ofcathepsin K complex. Accordingly, the invention features a kitcomprising an antibody that binds cathepsin K complex, and suitablebuffers. The probe or monoclonal antibody can be labeled to detectbinding to collagenolytic cathepsin K complex. In a preferredembodiment, the kit features a labeled antibody to cathepsin K complex.

[0019] Other objects and advantages will become apparent from a reviewof the ensuing detailed description taken in conjunction with thefollowing illustrative drawing.

BRIEF DESCRIPTION OF THE DRAWING

[0020]FIG. 1: Detection of CSA/Cat K complex formation and inhibition.CSA/Cat K complex formation and dissociation were tested in 100 mMsodium acetate buffer, pH 5.5 including 2 mM EDTA/DTT in a cuvette assyusing a Perkin-Elmer fluorimeter at excitation and emission wavelengthsof 300 nm and 604 nm, respectively. Curve 1: 120 nM Cat K, then 0.6μg/ml FL-CSA, and then 3 μg/ml dextran sulfate; Curve 2:,0.6 μg/mlFL-CSA, then 120 nM Cat K, and then 3 μg/ml dextran sulfate.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Before the present collagenolytically active cathepsin K complexmolecules, assay methodology, and treatment methodology are described,it is to be understood that this invention is not limited to particularassay methods, collagenolytically active cathepsin K complexes, or testcompounds and experimental conditions described, as such methods andcompounds may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention will be limited only the appended claims.

[0022] As used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural references unless thecontext clearly dictates otherwise. Thus for example, references to “acollagenolytically active cathepsin K complex” includes mixtures of suchcomplexes, reference to “the formulation” or “the method” includes oneor more formulations, methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

[0023] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to describe the methodsand/or materials in connection with which the publications are cited.

[0024] Definitions

[0025] By the phrase “collagenolytically active cathepsin K complex” ismeant a complex comprising a plurality of cathepsin K peptides inassociation with a plurality of glycosaminoglycan molecules whichexhibits a specific collagenolytic activity relative to free cathepsinK. “Specific collagenolytic activity” means an ability to hydrolyzetriple-helical collagen, e.g., type I or type II collagen.

[0026] A collagenolytically active cathepsin K complex may comprise amature cathepsin K peptide, a pro-peptide, pre-pro-peptide, analog,homolog, derivative, or variant thereof. The cathepsin K complex mayinclude any form of cathepsin K capable of forming an oligomeric complexwith specific collagenolytic activity, including, mature, pre- orpro-cathepsin K (see, for example, Shi et al. (1995) FEBS Lett.357:129-134, herein specifically incorporated by reference in itsentirety).

[0027] “Cathepsin K homolog” refers to a polypeptide that comprises anamino acid sequence similar to that of cathepsin K, but does notnecessarily possess a similar or identical function as the cathepsin K.“Cathepsin K ortholog” refers to a non-human polypeptide that (i)comprises an amino acid sequence similar to that of cathepsin K, and(ii) possesses a similar or identical function to that of cathepsin K.“Cathepsin K-related polypeptide” refers to a cathepsin K homolog, acathepsin K analog, a cathepsin K ortholog, or any combination thereof.“Cathepsin K derivative” refers to a polypeptide that comprises an aminoacid sequence of a cathepsin K polypeptide which has been altered by theintroduction of amino acid residue substitutions, deletions oradditions. The derivative polypeptide possesses a similar or identicalfunction as the cathepsin K polypeptide.

[0028] “Native” or “naturally occurring” cathepsin K complex means acollagenolytically active cathepsin K complex found in nature, forexample, in a mammal.

[0029] “Fragment” refers to a peptide or polypeptide comprising an aminoacid sequence of at least 5 amino acid residues (preferably, at least 10amino acid residues, at least 15 amino acid residues, at least 20 aminoacid residues, at least 25 amino acid residues, at least 40 amino acidresidues, at least 50 amino acid residues, at least 60 amino residues,at least 70 amino acid residues, at least 80 amino acid residues, atleast 90 amino acid residues, at least 100 amino acid residues, at least125 amino acid residues, at least 150 amino acid residues, at least 175amino acid residues, at least 200 amino acid residues, or at least 250amino acid residues) of the amino acid sequence of a cathepsin Kpolypeptide.

[0030] The term “substantially pure,” when referring to a polypeptide,means a polypeptide that is at least 60%, by weight, free from theproteins and naturally-occurring organic molecules with which it isnaturally associated. A substantially pure cathepsin K protein complexis at least 75%, more preferably at least 90%, and most preferably atleast 99%, by weight, human cathepsin K protein complex. A substantiallypure human cathepsin K protein complex can be obtained, for example, byextraction from a natural source; by expression of a recombinant nucleicacid encoding a human cathepsin K protein complex, or by chemicallysynthesizing the protein and adding the complexing agents. Purity can bemeasured by any appropriate method, e.g., column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

[0031] “Cathepsin K complex-associated disorders” or “cathepsin Kcomplex-mediated diseases” and such, mean disorders and diseases whichresult from excessive bone and cartilage erosion resulting from thecollagenolytic activity of the cathepsin K complex, or from insufficientcollagenolytic activity of the cathepsin K complex.

[0032] The term “modulation cathepsin K complex activity” meansmodulation (e.g., up or down regulation, enhancement or inhibition ofactivity, etc.) specifically of the collagenolytic activity of thecathepsin K complex of the invention, e.g., the triple-helical collagenhydrolyzing activity of the complex, and includes inhibitory orstimulatory effects. In one embodiment, the invention includes methodsfor screening reagents that inhibit or prevent cathepsin K complexformation, and thus decrease or prevent collagenolytic activity withouteffecting other biological activities of the free cathepsin K molecule.Such reagents are useful for the treatment or prevention of cathepsin Kprotein complex-mediated disorders, for example, osteoporosis, Paget'sdisease, and rheumatoid arthritis. The common feature of these diseasesis the excessive and irreversible degradation of extracellular bone andcartilage matrix.

[0033] As used herein, the term “therapeutic reagent” means any compoundor molecule that achieves the desired effect on a cathepsin Kcomplex-mediated disorder when administered to a subject in needthereof. A therapeutic reagent that “inhibits cathepsin K complexactivity” interferes with cathepsin K complex activity or cathepsin Kcomplex formation. For example, a therapeutic reagent can inhibitcollagenase breakdown by inhibiting the collagenolytic activity ofcathepsin K complex, or by inhibiting the formation of an activecathepsin K complex. A therapeutic reagent that “enhances cathepsin Kcomplex activity” increases the collagenolytic activity of the cathepsinK complex or promotes formation of the cathepsin K complex. A“therapeutically effective amount” is an amount of a reagent sufficientto decrease or prevent the symptoms associated with the cathepsin Kprotein complex-mediated disorder.

[0034] General Aspects of the Invention

[0035] The present invention described in detail below provides methodsfor the treatment and/or amelioration of disorders mediated specificallyby the collagenolytic activity of cathepsin K oligomeric complexes.

[0036] The instant invention is based, in part, on the discovery thatthe unique collagenolytic activity of cathepsin K requires the formationof an oligomeric cathepsin K complex from monomeric (free) cathepsin Kmolecules and specific glycosaminoglycans. Monomeric cathepsin K hasonly a minimal (if any) ability to degrade triple-helical collagens, buthas a high activity towards various non-collagenolytic substrates, e.g.,gelatin and synthetic peptide substrates. Cathepsin K peptide is able tocomplex with various glycosaminoglycans, but not all the complexesexhibit collagenolytic activity. The most potent glycosaminoglycansinvolved in formation of a productive collagenolytically active complexare bone-and cartilage-resident chondroitin and keratan sulfates.

[0037] This discovery makes possible a completely new strategy toselectively inhibit or enhance the pharmaceutically relevantcollagenolytic activity of cathepsin K towards triple-helical collagenswithout interfering with other activities of the enzyme. Compoundsinterfering with the formation of functionally active collagenolyticcathepsin K complex will lead to a selective loss of the cathepsin Kcomplex collagenase activity, while retaining the activity of freecathepsin K towards most non-collagen substrates. Therefore, the designof selective complex formation inhibitors is expected to avoidpotentially negative side effects of classical site-directed inhibitors,as well as avoid difficulties in the design of highly specificinhibitors due to redundancy of closely related cathepsins with similaractive sites.

[0038] Collagenolytically active cathepsin K is a complex comprised offive cathepsin K peptides and five chondroitin sulfate (CS) molecules,with a composite molecular weight of 275 kD. Electron microscopyanalysis revealed a pentameric ring structure of 120-130 Å in diameterwith a central pore of 25-30 Å sufficient to accommodate helicalcollagen trimers with a diameter of 15 Å. As is described more fullybelow, inhibition of complex formation led to the loss of collagenolyticactivity but did not affect the activity of the enzyme towards gelatinand synthetic substrates.

[0039] Screening Assays

[0040] The invention provides methods for identifying agents (e.g.,chemical compounds, carbohydrates, proteins, peptides, or nucleotides)that have a stimulatory or inhibitory effect on the formation oractivity of a collagenolytically active cathepsin K complex. Theinvention also provides methods of identifying agents, candidatecompounds or test compounds that specifically bind to a cathepsin Kcomplex. Examples of agents, candidate compounds or test compoundsinclude, but are not limited to, nucleic acids (e.g., DNA and RNA),carbohydrates, lipids, proteins, peptides, peptidomimetics, smallmolecules and other drugs. Agents can be obtained using any of thenumerous suitable approaches in combinatorial library methods known inthe art, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the “one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, 1997, Anticancer Drug Des.12:145; U.S. Pat. No. 5,738,996; and U.S. Pat. No. 5,807,683, each ofwhich is incorporated herein in its entirety by reference).

[0041] In one embodiment, agents that interact with (i.e., bind to) acathepsin K complex comprising cathepsin K polypeptide, a cathepsin Kpeptide fragment (e.g. a functionally active fragment), or a cathepsinK-related polypeptide, are identified in a cell-based assay system. Inaccordance with this embodiment, cells expressing a cathepsin K complexcomprising a cathepsin K peptide or polypeptide, a fragment thereof, ora cathepsin K-related polypeptide, are contacted with a candidatecompound or a control compound and the ability of the candidate compoundto interact with the cathepsin K complex is determined. If desired, thisassay may be used to screen a plurality (e.g., a library) of candidatecompounds. The cell, for example, can be of prokaryotic origin (e.g., E.coli) or eukaryotic origin (e.g., yeast or mammalian). Further, thecells can express the cathepsin K peptide or polypeptide, fragment, orrelated polypeptide thereof, endogenously or be genetically engineeredto express the cathepsin K peptide or polypeptide, fragment, or relatedpolypeptide thereof. In some embodiments, the cathepsin K peptide orpolypeptide, fragment, or related polypeptide thereof or the candidatecompound is labeled, for example with a radioactive label (such as ³²P,³⁵S or ¹²⁵I or a fluorescent label (such as fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeor fluorescamine) to enable detection of an interaction between acathepsin K complex and a candidate compound. The ability of thecandidate compound to interact directly or indirectly with the cathepsinK complex can be determined by methods known to those of skill in theart. For example, the interaction can be determined by flow cytometry, ascintillation assay, immunoprecipitation or western blot analysis.

[0042] In another embodiment, agents interact with (i.e., bind to) acathepsin K complex in a cell-free assay system. In accordance with thisembodiment, a cathepsin K complex is contacted with a candidate compoundor a control compound and the ability of the candidate compound tointeract with the cathepsin K complex is determined. If desired, thisassay may be used to screen a plurality (e.g. a library) of candidatecompounds. In specific embodiments, the cathepsin K complex is firstimmobilized, by, for example, contacting the cathepsin K complex with animmobilized antibody which specifically recognizes and binds it, or bycontacting a purified preparation of the cathepsin K complex with asurface designed to bind proteins. The cathepsin K complex may bepartially or completely purified (e.g., partially or completely free ofother polypeptides) or part of a cell lysate. The ability of thecandidate compound to interact with the cathepsin K complex can be canbe determined by methods known to those of skill in the art.

[0043] In another embodiment, a cell-based assay system is used toidentify agents that bind to or modulate the activity of the cathepsin Kcomplex, or a biologically active portion thereof, which is responsiblefor the degradation of triple-helical collagen. In a primary screen, aplurality (e.g., a library) of compounds are contacted with cells thatnaturally or recombinantly express components which form anenzymatically active collagenolytic cathepsin K complex in order toidentify compounds that modulate the formation of the complex molecule.The ability of the candidate compound to modulate the formation of thecathepsin K complex can be determined by methods known to those of skillin the art, including without limitation, flow cytometry, ascintillation assay, immunoprecipitation and western blot analysis.

[0044] In another embodiment, agents that competitively interact with(i.e., bind to) a cathepsin K complex are identified in a competitivebinding assay. In accordance with this embodiment, cells expressingcomponents of the cathepsin K complex able to form thecollagenolytically active complex are contacted with a candidatecompound and a compound known to interact with the cathepsin K complexor prevent the formation of an active cathepsin K complex; the abilityof the candidate compound to competitively interact with the cathepsin Kcomplex or to competitively prevent formation of the cathepsin K complexis then determined. Alternatively, agents that competitively interactwith (i.e., bind to) a cathepsin K complex or competitively prevent theformation of an collagenolytically active cathepsin K complex areidentified in a cell-free assay system by contacting the cathepsin Kcomplex or components able to form an active cathepsin K complex with acandidate compound and a compound known to interact with or prevent theformation of the cathepsin K complex. As stated above, the ability ofthe candidate compound to interact with a cathepsin K complex or preventthe formation of an active cathepsin K complex can be determined bymethods known to those of skill in the art. These assays, whethercell-based or cell-free, can be used to screen a plurality (e.g., alibrary) of candidate compounds.

[0045] In another embodiment, agents that modulate (i.e., up-regulate ordown-regulate) the formation of an collagenolytic cathepsin K complex orthe collagenolytic activity of an existing cathepsin K complex areidentified by contacting cells (e.g., cells of prokaryotic origin oreukaryotic origin) expressing the components capable of forming anenzymatically active collagenolytic cathepsin K complex with a candidatecompound or a control compound (e.g. phosphate buffered saline (PBS))and determining the formation or activity of the cathepsin K complex.The level of active complex formation or complex activity in thepresence of the candidate compound is compared to the level of formationor activity in the absence of the candidate compound (e.g., in thepresence of a control compound). The candidate compound can then beidentified as a modulator of the expression of the cathepsin K complexbased on this comparison. For example, when presence of an enzymaticallyactive cathepsin K complex is significantly greater in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as a stimulator of complex formation and/or an enhancer ofcomplex activity. Alternatively, when presence of an enzymaticallyactive cathepsin K complex is significantly less in the presence of thecandidate compound than in its absence, the candidate compound isidentified as an inhibitor of complex formation and/or inhibitor ofcomplex activity.

[0046] In another embodiment, agents that modulate the activity of anenzymatically active collagenolytic cathepsin K complex are identifiedby contacting a preparation containing the complex, or cells (e.g.,prokaryotic or eukaryotic cells) forming an enzymatically activecollagenolytic cathepsin K complex with a test compound or a controlcompound and determining the ability of the test compound to modulate(e.g., stimulate or inhibit) the activity of the cathepsin K complex.The activity of the cathepsin K complex can be assessed in a number ofways, including by, e.g., determining hydrolysis of triple-helicalcollagen.

[0047] In another embodiment, agents that modulate (i.e., up-regulate ordown-regulate) the formation, activity or both, of a cathepsin K complexare identified in an animal model. Examples of suitable animals include,but are not limited to, mice, rats, rabbits, monkeys, guinea pigs, dogsand cats. Preferably, the animal used represents a model of a cathepsinK complex-associated disorder, e.g., osteoporosis.

[0048] In accordance with this embodiment, the test compound or acontrol compound is administered (e.g., orally, rectally or parenterallysuch as intraperitoneally or intravenously) to a suitable animal and theeffect on the formation, activity or both formation and activity of thecathepsin K complex is determined, or the effect on a cathepsin Kcomplex target cell is determined. Changes in the activity of acathepsin K complex can be assessed by any suitable method describedabove, based on the present description.

[0049] This invention further provides novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

[0050] Therapeutic Uses of the Invention

[0051] The invention provides for treatment or prevention of variousdiseases and disorders by administration of a therapeutic agent. Suchagents include but are not limited to: agents which promote or preventformation of the enzymatically active collagenolytic cathepsin Kcomplex, agents which modulate the activity of enzymatically activecollagenolytic cathepsin K complex, agents able to act as antagonists oragonists of the enzyrnatically active collagenolytic cathepsin Kcomplex, and the enzymatically active collagenolytic cathepsin K complexitself, and related analogs, derivatives, and fragments thereof;antibodies to the foregoing.

[0052] In one embodiment wherein inhibition of the enzymatically activecollagenolytic cathepsin K complex is desirable, one or more antibodieseach specifically binding to the cathepsin K complex are administeredalone or in combination with one or more additional therapeuticcompounds or treatments. Preferably, a biological product such as anantibody is allogeneic to the subject to which it is administered. In apreferred embodiment, a human cathepsin K complex is administered to ahuman subject for therapy (e.g. to ameliorate symptoms or to retardonset or progression) or prophylaxis.

[0053] Assays For Therapeutic or Prophylactic Compounds

[0054] The present invention also provides assays for use in discoveryof pharmaceutical products in order to identify or verify the efficacyof compounds for treatment or prevention of cathepsin K complex-mediateddiseases. In one embodiment, agents can be assayed for their ability toreduce bone resorption activity in a subject suffering from osteoporosistowards levels found in subjects free from any osteoporosis diseases orto produce similar changes in experimental animal models ofosteoporosis. Compounds able to reduce the collagenolytic activity ofcathepsin K complex levels in a subject suffering from a disorderrelated to excessive bone resorption in a subject towards healthy levelsfound in subjects free from osteoporosis, or able to produce similarchanges in experimental animal models of osteoporosis can be used aslead compounds for further drug discovery, or used therapeutically.

[0055] In various embodiments, in vitro assays can be carried out withcells representative of cell types involved in a subject's disorder, todetermine if a compound has a desired effect upon such cell types. Inone embodiment, the cells are skin fibroblast cells, and the conditionis undesirable wrinkling or aging due to age, smoking, sun-exposure, orother conditions.

[0056] Compounds for use in therapy can be tested in suitable animalmodel systems prior to testing in humans, including but not limited torats, mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing,prior to administration to humans, any animal model system known in theart may be used. In one embodiment, test compounds that modulate theformation or activity of enzymatically active collagenolytic cathepsin Kcomplex are identified in non-human animals (e.g., mice, rats, monkeys,rabbits, and guinea pigs), preferably non-human animal models forcathepsin K complex-associated disorders. In accordance with thisembodiment, a test compound or a control compound is administered to theanimals, and the effect of the test compound on collagenolytic cathepsinK complex levels or activity is determined. A test compound that altersthe level or activity of collagenolytic cathepsin K complex can beidentified by comparing the level of the selected collagenolyticcathepsin K complex in an animal or group of animals treated with a testcompound with the level of the collagenolytic cathepsin K complex in ananimal or group of animals treated with a control compound.

[0057] In another embodiment, test compounds that modulate the activityof collagenolytic cathepsin K complex are identified in non-humananimals (e.g., mice, rats, monkeys, rabbits, and guinea pigs),preferably non-human animal models for collagenolytic cathepsin Kcomplex-mediated disorders. In accordance with this embodiment, a testcompound or a control compound is administered to the animals, and theeffect of a test compound on the activity of collagenolytic cathepsin Kcomplex is determined. A test compound that alters the activity ofcollagenolytic cathepsin K complex can be identified by assaying animalstreated with a control compound and animals treated with the testcompound.

[0058] In yet another embodiment, test compounds that modulate the levelor activity of collagenolytic cathepsin K complex are identified inhuman subjects having a cathepsin K complex-associated disorder. Inaccordance with this embodiment, a test compound or a control compoundis administered to the human subject, and the effect of a test compoundon collagenolytic cathepsin K complex activity, or bone resorption isdetermined by methods known in the art.

[0059] Therapeutic and Prophylactic Compositions and Their Use

[0060] The invention provides methods of treatment comprisingadministering to a subject an effective amount of an agent of theinvention. In a preferred aspect, the compound is substantially purified(e.g., substantially free from substances that limit its effect orproduce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human. In one specific embodiment, a non-human mammal is thesubject. In another specific embodiment, a human mammal is the subject.

[0061] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid are described above;additional appropriate formulations and routes of administration aredescribed below. Various delivery systems are known and can be used toadminister a compound of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the compound, receptor-mediated endocytosis (see, e.g., Wuand Wu, 1987, J. Biol. Chem. 262:44294432), construction of a nucleicacid as part of a retroviral or other vector, etc. Methods ofintroduction can be enteral or parenteral and include but are notlimited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The compounds maybe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. In a specificembodiment, it may be desirable to administer the pharmaceuticalcompositions of the invention locally to the area in need of treatment;any suitable method known to the art may be used.

[0062] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of anagent, and a pharmaceutically acceptable carrier. In a particularembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the subject. Theformulation should suit the mode of administration.

[0063] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lidocaine toease pain at the site of the injection. Generally, the ingredients aresupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0064] The amount of the compound of the invention which will beeffective in the treatment of cathepsin K complex-mediated disorders canbe determined by standard clinical techniques based on the presentdescription. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each subject's circumstances.However, suitable dosage ranges for intravenous administration aregenerally about 20-500 micrograms of active compound per kilogram bodyweight. Suitable dosage ranges for intranasal administration aregenerally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems.

[0065] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflects(a) approval by the agency of manufacture, use or sale for humanadministration, (b) directions for use, or both.

[0066] Collagenolytically Active Cathepsin K Complex

[0067] The experiments described below demonstrate that bone andcartilage-resident glycosaminoglycans (GAGs) dramatically andspecifically enhance the degradation of interstitial collagens of typesI and II by cathepsin K. This effect is not seen with cathepsin L ormatrix metalloproteinase I (Li (2000) Biochemistry 39:529-536).Hydrolysis of type I collagen by cathepsin K was shown to be dependenton the presence of chondroitin 4-sulfate (CSA), and specificallyenhanced by increasing concentrations of CSA (Example 1).

[0068] Analysis of commercially available type I and II collagen samplesrevealed that contaminations with GAGs were responsible for thepreviously reported cathepsin K-mediated collagen degradation in theapparent absence of GAGs (Kafienah et al. (1998) Biochem J. 331:727-732;Garnero et al. (1998) J Biol Chem 273:32347-32352). Reduction of the GAGcontent in collagen samples by chondroitinase ABC treatment resulted ina significant reduction of the collagenolytic activity of cathepsin Ktowards type I collagen. This finding indicated that the presence ofGAGs is essential for the collagen-degrading activity of cathepsin K.

[0069] Size-exclusion chromatography of purified recombinant cathepsin Kand CSA mixtures revealed a proteolytically active, high molecularweight complex whose mass varied from 200-310 kD depending on themolecular mass fraction of CSA used (17 to 30 kD determined by dynamiclight scattering) (Example 2).

[0070] When cathepsin K/CSA mixtures were eluted in the presence of 300mM NaCl, cathepsin K activity corresponded to a peak of 25 kD identicalto the elution peak obtained when cathepsin K was applied without CSA tothe gel filtration column. This indicates that high salt concentrationinhibits the formation of cathepsin K complexes. The absence or presenceof NaCl had only a minor effect on the k_(cat)/K_(m). value of cathepsinK on the hydrolysis of the fluorigenic dipeptide substrate, Z-LR-MCA,whereas the presence of 0.15% CSA doubled the specific activity (Table1).

[0071] Complex formation was also detected in a electrophoretic mobilityshift assay using [¹²⁵]DCG-04 labeled cathepsin K in the presence ofCSA. Cathepsin K/CSA complexes migrated to the anode whereas in theabsence of CSA cathepsin K migrated to the cathode. In the presence ofNaCl, the complex dissociated and cathepsin K migrated identically tothe protease sample in the absence of CSA.

[0072] Using the mobility shift assay, the ratio between [^(125])DCG-04labeled cathepsin K and CSA in the complex was determined. The plot ofcathepsin K complex/CSA vs cathepsin K concentration resulted in asaturation curve reaching a plateau at a 1:1 ratio between cathepsin Kand CSA.

[0073] The molecular mass of the cathepsin K/CSA complex in the presenceof 30 kD CSA was determined by dynamic light scattering (DLS) andanalytical ultracentrifugation. Measurements by DLS yielded in amolecular mass of 274±12 kD and the value obtained byultracentrifugation was 284 kD Based on the molecular mass of thecomplex and the ratio of 1:1 between cathepsin K and CSA, the complexwas determined to have the following stoichiometry: Cat K₅ CSA₅ with amolecular mass of 275 kD (using molecular masses for CSA, 29.8 kD andCat K, 24.7 kD as determined by DLS). The hydrodynamic radius of thecomplex was determined by DLS to be 65.3±1.4 Å. Stoichiometry andhydrodynamic radius of the complex suggest a ring structure containingfive cathepsin K molecules connected via five positively charged CSAmolecules.

[0074] Electron microscopic examination supports the predicted5-membered ring structure (not shown). The ring structure has a diameterof 120-130 Å with a central pore measuring 25-30 Å. This pore wouldallow access for linear triple helical collagen molecules with adiameter of 15 Å.

[0075] To determine if complex formation is required for thecollagenolytic activity of cathepsin K, type I collagen was incubatedwith free cathepsin K or with cathepsin K/CSA complexes at 28 C and 37 Cin the presence or absence of 300 mM NaCl. In the absence of NaCl,collagen is completely degraded by the protease/CSA complex whereas inthe presence of NaCl neither cathepsin K alone nor in a mixture with CSAshowed any degradation of the extracellular matrix protein. Thissuggested that triple helical type I collagen is resistant towards theactivity of monomeric cathepsin K even at 37 C. The results showed that300 mM NaCl inhibits oligomerization of cathepsin K in the presence ofCSA. NaCl had no effect on the degradation of denatured type I collagen(gelatin) which lost its triple helical structure thus indicating thatcomplex formation is not required for cathepsin K-catalyzed degradationof non-collagenous protein substrates. The slightly increaseddegradation of gelatin in the presence of CSA can be attributed to thestabilizing effect of GAGs on cathepsin K activity during long-timeincubation periods as previously reported (Li (2000) supra). Therefore,the collagenolytic activity of cathepsin K only depends on complexformation with CSA but not on cathepsin K stability or interactionsbetween CSA and collagen.

[0076] To date, 10 mutations in the cathepsin K gene of pycnodysostosispatients have been described (Gelb et al. (2001) Science 273:1236-1238).With the exception of mutant Y212C, all other mutations characterized todate represent nonsense or missense mutations resulting in unstableproteins or no protein (Hou et al. (1999) J Clin Invest 103:731-738).The carrier of the Y212C mutation is hetero-allelic and has in additionthe non-sense mutation, R-241X. Whereas both heterozygous parents do notexhibit the pycnodysostosis phenotype, the double allelic mutations inthe child displays the typical disease characteristics such as shortstature, skeletal, craniofacial and dental abnormalities.

[0077] We have recently shown that mutant Y212C lacked collagenaseactivity but retained a strong gelatinase activity and significantactivities towards synthetic peptide substrates (Hou et al. (1999)supra). We concluded that pycnodysostosis is specifically caused by thedeficiency of the collagenolytic activity of cathepsin K and not apriori by the absence of cathepsin K protein and activity.

[0078] In the experiments below, it is demonstrated that the Y212Cmutant is unable to form a collagenolytically active complex with CSA.Neither at low CSA concentrations (0.2 μg/ml, equal to the concentrationof CSA in commercial collagen preparations) nor at high concentrations(1000 μg/ml) any complex formation between Y212C and CSA was observed.In contrast, wild-type cathepsin K formed complexes at both CSAconcentrations. The Y212C mutant did not reveal any collagenase activityneither in the presence of or absence of CSA. Thus the lack of complexformation by Y212C explains the inability of this mutant form tohydrolyze native collagen. In contrast, the gelatinase activities ofwild-type cathepsin K and mutant protease in the presence of CSA werecomparable supporting the hypothesis that complex formation isexclusively required for the collagenase activity of cathepsin K.Complex formation in the presence of CSA is unique for cathepsin K.Closely related cysteine proteases such as cathepsins L, B, and S areunable to form complexes which may explain their inability to cleave inthe triple-helical region of interstitial collagens (data not shown).

[0079] Examples 15-16 below describe a method for detectingchondroitin4-sulfate (CSA)/Cat K complex formation, and application to ahigh-through put screening method for identifying agents capable ofinhibiting CSA/Cat K complex formation. Example 17 reports the resultsof mutational studies designed to examine the sites of glycosaminoglycanand cathepsin K interaction.

EXAMPLES

[0080] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the collagenolytically active cathepsin K complex,assay, screening, and therapeutic methods of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Hydrolysis of Type I Collagen by Cathepsin K

[0081] 30 μg of cathepsin K (cat K) were applied to a Superdex 200column, and eluded with 100 mM sodium acetate buffer, pH 5.5, 1 mMEDTA/DTT, without and with 0.3 M NaCl, cathepsin K degradation fragmentseluted as peaks present between 20-28 ml elution volume. Free cathepsinK eluded at Ve 17 ml in the presence of 0.3 M NaCl, whereas it binds tothe column in the absence of NaCl. Soluble type I bovine collagen wasdigested with 800 nM cathepsin K at 28° C., in 100 mM sodium acetatebuffer pH 5.5, containing 2.5 mM EDTA/DTT. The reaction was stopped bythe addition of 10 μM E-64 after various time periods and the sampleswere analyzed by 4-20% SDS-Page. The results showed that free cathepsinK displays a weak degradation of type I collagen. Degradation productsare likely derived from partially denatured type I collagen. Solubletype I bovine collagen (United States Biochemicals, Cleveland, OH) wasdigested with 800 nM human recombinant cathepsin K in the presence ofincreasing concentrations of chondroitin 4-sulfate (CSA) at 28° C., in100 mM sodium acetate buffer, pH 5.5, containing 2.5 mM EDTA/DTT. Thereaction was stopped by the addition of 10 μM E-64 after 8 h ofincubation and the samples were analyzed by 4-20% SDS-polyacrylarnideelectrophoresis.

[0082] 0.2 μg/ml was determined to be the endogenous CSA concentrationin commercial type I collagen preparations. The concentration of GAGsdropped to 0.1 μg/ml after depletion of CSA by chondroitinase ABCtreatment, accompanied by a strongly reduced ability of cathepsin K tocleave triple helical collagen. At concentrations of CSA at 10 μg/ml andhigher, saturation of the complex formation was reached and the cleavageefficacy of type I collagen by cathepsin K/CSA complexes exhibited itsmaximum. Residual GAGs from commercial type I collagen were removed bytreatment with chondroitinase ABC. 100 μl 8 mg/ml collagen (USB,Cleveland, Ohio) in 100 mM sodium acetate buffer, pH 5.5 were incubatedwith 50 mU chondroitinase ABC (Sigma, St. Louis, Mo.) at 25 C for 4 h.Residual GAG content was determined as followed: 8 mg/mi collagen wereheated at 70° C. for 20 min and then incubated with 1 mg/ml pepsin at 25C, pH 3.7 for 2 h. GAG concentration was measured using the Blyscanglycosaminoglycan assay kit (Biocolor Ltd., Ireland).

Example 2 Cathepsin K/CSA Complex

[0083] 30 μg of cathepsin K were preincubated with elution buffercontaining 0.15% medium molecular size fraction (Ve 58.46 ml) ofchondroitin 4-sulfate (CSA) for 20 min, and then applied to a Superdex200 column. The elution buffer was 100 mM sodium acetate, pH 5.5, 1 mMEDTA/DTT. The CSA/cathepsin K complex was eluded at Ve of 11 ml.

[0084] A mixture of cathepsin K and CSA (30 kD fraction) wasfractionated by Superdex 200 gel filtration. In the absence of NaCl, thecathepsin K collagenase activity was associated with a high molecularweight complex of approximately 310 kD.

[0085] In the presence of 300 mM NaCl, cathepsin K collagenase activitywas eluted from the same mixture as a monomeric protease with anapparent molecular mass of 24 kD. 40 μg of purified recombinant humancathepsin K in the presence of 0.1% CSA were applied to a column andeluted either in the presence or absence of 300 mM NaCl with 100 mMsodium acetate buffer, pH 5.5 containing 1 mM EDTA/ DTT. The proteinelution was recorded at OD₂₈₀ nm and fractions were assayed for thehydrolysis of the cathepsin K substrate Z-Leu-Arg-MCA. 50 ng of[¹²⁵]DCG-04 labeled cathepsin K were preincubated in 100 mM sodiumacetate buffer, pH 5.0 containing 0.5 mM EDTA/DTT in the presence orabsence of 0.1% of CSA, respectively, for 20 min and then mixed at 37°C. with non-reducing protein loading buffer and preheated agarose gel(37 C; final concentration at 0.4%). The sample was loaded into the wellof a 0.5% agarose gel and separated at room temperature at 55 V, 250 mAfor 30 min in a running buffer containing 125 mM sodium acetate, pH5.0,0.5 mM EDTA, 1 mM DTT, 40 mM NaCl, and 0.1% Chaps. The gel was driedand subsequently exposed to an X-ray film. The conditions for lane 3were modified by the addition of 300 mM NaCl into the agarose gel andrunning buffer. Table 1 shows the results of collagen hydrolysis of thefluorigenic dipeptide substrate Z-LR-MCA by cathepsin K in the absenceand presence of 0.15% CSA and 300 mM NaCl. Activity assays wereperformed using Z-LR-MCA as substrate in 100 mM sodium acetate buffer,pH 5.5, containing 2.5 mM EDTA/DTT and 0.5 nM recombinant humancathepsin K. TABLE 1 Condition k_(cat)/K_(m) (M⁻¹s⁻¹) × 106 — 1.199 ×10⁵ GSA 2.404 × 10⁵ NaCl 0.982 × 10⁵ CSA/NaCl 1.615 × 10⁵

Example 3 Human Cathepsin K Forms Complexes with CSA at a Ratio of 1:1

[0086] Increasing amounts of [¹²⁵]DCG-04 labeled cathepsin K (0 to 1425pmoles) were separated in a 0.5% agarose gel containing 292 pMol of CSA(30 kD fraction) as described above. After exposure of the dried gel toan X-ray film, the signal of free cathepsin K was densitometricallydetermined and the amount of cathepsin K in the complex was calculated(cat K_(compley)=cat K_(total)−cat K_(free)). Plotting the ratio catK_(complex)/CSA vs. the cat K_(total) a curve was obtained reaching aplateau at 1 for the catK_(complex)/CSA ratio.

Example 4 Triple-Helical Type I Collagen Degradation by Cathepsin K/CSA

[0087] Soluble type I bovine collagen was digested with 800 nM purifiedrecombinant human cathepsin K and gelatin (soluble type I collagen washeated for 20 min at 70 C) with 0.5 nM cathepsin K at 28° C. and 37 C,in 100 mM sodium acetate buffer, pH 5.5, containing 2.5 mM EDTA/DTT.Digestion experiments were carried out in the presence or absence of 300mM NaCl and 0.15% CSA. The reaction was stopped by the addition of 10 μME-64 after 8 h of incubation and the samples were analyzed by 4-20%SDS-polyacrylamide electrophoresis.

Example 5 Elution Profile for Cathepsin K Complexes Formed in thePresence of Negatively Charged Polymers

[0088] 30 μg of cathepsin K were preincubated with elution buffercontaining 0.15% low molecular fraction (V3 67.2 ml) of dermatan sulfate(DS) 0.15% 2-15 kD poly-D-Glu, or 0.15% 10 kD dextran sulfate for 20min, and then applied to a Superdex 200 column. The elution buffer was100 mM sodium acetate, pH 5.5, containing 1 mM EDTA/DTT. Severalcathepsin K complexes were formed. DS/Cat K complex was present at a Veof 14 ml, poly-D-Glu/Cat K at 14.2 ml, dextran sulfate/Cat K complex at13.5 ml. These results support the ability of cathepsin K to formcomplexes with negatively charged linear polymers.

[0089] 15 μg of cathepsin K were preincubated with running buffer in thepresence or the absence of 0.15% high molecular weight fraction (Ve44.06 ml) of CSA, 0.15% heparin (6 kD), 0.15% keratan sulfate (KS),0.15% dermatan sulfate (DS), and 0.15% C-6S, respectively, for 20 min.Samples were subsequently mixed with non-reducing protein sample buffer,DNA dye, and melted agarose gel (melted at 37 C; 0.4% finalconcentration) at 37 C and embedded into a pre-made wells of a 0.5%agarose gel (running conditions: 25° C.; 55 V, 250 mA for 30 min). Thegel was stained with toluidine blue for glycosaminoglycans and coomassieblue for protein. The running buffer was 125 mM sodium acetate, pH5.0,0.5 mM EDTA, 1 mM DTT, 40 mM NaCl, and 0.1% Chaps. The results showthat complexes CSA/cathepsin K, heparin/cathepsin K, KS/cathepsin K,DS/Cat K, and C-6S/Cat K, were found in the presence of CSA, heparin,KS, DS, and C-6S, respectively.

Example 6 Gelatin and Type I Collagen Degradation

[0090] Gelatin and soluble type I bovine collagen were digested with 800nM and 1 nM cathepsin K, respectively, at 28° C., 100 mM sodium acetatebuffer pH 5.5, 2.5 mM EDTA/DTT, with/without 0.15% CSA, 0.15% dextransulfate, and 0.15% poly-D-Glu. The reactions were stopped by theaddition of 10 μM E-64 and the samples were analyzed by 4-20% SDS-Page.The results show that cathepsin K formed complexes with CSA, dextransulfate, and poly-D-Glu. The complexes formed with dextran sulfate andpoly-D-Glu did not exhibit collagenase activity, but were active againstdenatured collagen gelatin. All complexes stabilize cathepsin Kactivity, such that the cumulative gelatinolytic activity (Li et al.(2000) supra) was higher than that of free cathepsin K. Only theCSA/cathepsin K complex displayed activity towards native triple-helicalcollagen, indicating that the strong collagenolytic activity of theCSA/cathepsin K complex is not caused by increased enzyme stability butis an intrinsic feature of the complex structure.

Example 7 Inhibition of CSA/Cathepsin K Complex Formation by DextranSulfate and Poly-D-Glu

[0091] 15 (g of cathepsin K were preincubated with running buffer in thepresence or absence of 0.15% high molecular weight fraction (Ve 44.06ml) of CSA, 0.15% dextran sulfate (10 kD), and 0.15% poly-D-glu (2-15kD) for 20 min; 8 (g of free cathepsin K was used as a control. Sampleswere subsequently mixed with non-reducing protein sample buffer, DNAdye, and melted agarose gel (melted at 37° C.; 0.4% final concentration)at 37° C. and embedded into a pre-made wells of a 0.5% agarose gel(running conditions: 25 oC; 55 V, 250 mA for 30 min). The gel wasstained with toluidine blue for glycosaminoglycans and coomassie bluefor protein. The running buffer was 125 mM sodium acetate, pH 5.0, 0.5mM EDTA, 1 mM DTT, 40 mM NaCl, and 0.1% Chaps. In the presence ofdextran sulfate or poly-D-Glu, the formation of the CSA/cathepsin Kcomplex was blocked by the competitive formation of dextransulfate/cathepsin K or poly-D-glu/cathepsin K complexes. This suggeststhat both dextran sulfate and poly-D-glu bind with a higher affinity tocathepsin K than CSA.

Example 8 Elution Profile for CSA/Cathepsin K Complex in the Presence of0.15% Dextran Sulfate

[0092] 30 μg of cathepsin K were preincubated with elution buffercontaining 0.15% medium molecular weight fraction (58.46) of CSA and0.15% dextran sulfate (10 kD) for 20 min, and then applied to a Superdex200 column. The elution buffer was 100 mM sodium acetate, pH 5.5, 1 mMEDTA/DTT. Instead of the CSA/cathepsin K complex (at Ve of 11 ml) acathepsin Kidextran sulfate complex was formed (Ve of 13.5 ml). Solubletype I bovine collagen was digested with 800 nM cathepsin K at 28° C.,100 mM sodium acetate buffer pH 5.5, containing 0.15% CSA, 0.15% ofdextran sulfate, and 2.5 mM EDTA/DTT. The reaction was stopped by theaddition of 10 μM E-64 and the samples were analyzed by 4-20% SDS-Page.The results establish that 0.15% dextrane sulfate prevented theformation of a cathepsin K/CSA complex, and thus prevents thedegradation of type I collagen. Dextran sulfates competes with CSA inthe complex formation with cathepsin K and forms a complex lackingcollagenolytic activity.

Example 9 Elution Profile for CSA/Cathepsin K Complex at 0.15%Poly-D-Glu

[0093] 30 g of cathepsin K were preincubated with elution buffercontaining 0.15% medium molecular weight fraction (Ve 58.46 ml) of CSAand 0.15% poly-D-glu (2-15 kDa) for 20 min, and then applied to aSuperdex 200 column. The elution buffer was 100 mM sodium acetate, pH5.5, 1 mM EDTA/DTT. The CSA/cathepsin K complex (Ve of 11 ml) was notformed, but a cathepsin K/poly-D-glu complex eluted at Ve of 14.2 ml.Soluble type I bovine collagen was digested with 800 nM cathepsin K at28° C., 100 mM sodium acetate buffer pH 5.5, containing 0.15% CSA, 0.15%poly-D-glu, and 2.5 mM EDTA/DTT. The reaction was stopped by theaddition of 10 μM E-64 and the samples were analyzed by 4-20% SDS-Page.

[0094] The results established that 0.15% poly-D-glu prevented theformation of a cathepsin K/CSA complex, and thus prevents thedegradation of type I collagen. Poly-D-glu competes with CSA in thecomplex formation with cathepsin K and forms a complex lackingcollagenolytic activity.

Example 10 Effect of a 30-mer Oligonucleotide on the Formation of aCSA/Cathepsin K Complex and its Collagenolytic Activity

[0095] 15 μg of cathepsin K were preincubated with running buffer in thepresence or the absence of 0.15% high molecular weight fraction (44.06)of CSA and 0.3% oligonucleotide (11 kDa) for 20 min. Samples weresubsequently mixed with non-reducing protein sample buffer, DNA dye, andmelted agarose gel (melted at 37 C; 0.4% final concentration) at 37 Cand embedded into a pre-made wells of a 0.5% agarose gel (runningconditions: 25° C.; 55 V, 250 mA for 30 min). The gel was stained withtoluidine blue for glycosaminoglycans and coomassie blue for protein.The running buffer was 125 mM sodium acetate, pH 5.0, 0.5 mM EDTA, 1 mMDTT, 40 mM NaCl, and 0.1% Chaps.

[0096] In the presence of both CSA and oligonucleotide, anoligonucleotide/cathepsin K complex was formed instead of theCSA/cathepsin K complex. Soluble type I bovine collagen was digestedwith 800 nM cathepsin K at 28° C., 100 mM sodium acetate buffer pH 5.5,containing 0.15% CSA, 0.3% of oligonucleotide, and 2.5 mM EDTA/DTT. Thereaction was stopped by the addition of 10 μM E-64 and the samples wereanalyzed by 4-20% SDS-Page. 0.3% of a 30-mer oligonucleotide preventedthe formation of a cathepsin K/CSA complex, and thus prevents thedegradation of type I collagen. The oligomer-nucleotide competed withCSA in the complex formation with cathepsin K and forms a complexlacking collagenolytic activity.

Example 11 Effect of CSA Disaccharide on Free Cathepsin K andCSA/Cathepsin K Complex Formation

[0097] 30 μg of cathepsin K were preincubated with elution buffercontaining 0.15% CSA disaccharide without or with 0.15% medium molecularweight fraction (Ve 58.46 ml) of CSA for 20 min, and then applied to aSuperdex 200 column. The elution buffer was 100 mM sodium acetate, pH5.5, 1 mM EDTA/DTT. Soluble type I bovine collagen was digested with 800nM cathepsin K at 28° C., 100 mM sodium acetate buffer pH 5.5,containing 0.15% CSA, 0.15% CSA disaccharide, and 2.5 mM EDTA/DTT. Thereaction was stopped by the addition of 10 μM E-64 and the samples wereanalyzed by 4-20% SDS-Page.

[0098] CSA disaccharides were unable to generate high-molecularcomplexes with cathepsin K and did not interfere with complex formationin the presence CSA. Thus, the collagenolytic activity of theCSA/cathepsin K complex was identical in the presence of CSAdisaccharide. This suggests that chondroitin disaccharide-4S does notbind with cathepsin K, and neither affected the CSA/cathepsin K complexformation or the collagenolytic activity of the complex.

Example 12 Effect of Dextran on Free Cathepsin K and CSA/Cathepsin KComplex Formation

[0099] 30 μg of cathepsin K were preincubated with elution buffercontaining 0.15% dextran (18 kD) without or with 0.15% medium molecularweight fraction (58.46) of CSA for 20 min, and then applied to aSuperdex 200 column. The elution buffer was 100 mM sodium acetate, pH5.5, 1 mM EDTA/DTT. The results showed that dextran did not form a highmolecular weight complex with cathepsin K. Further, the formation of theCSA/cathepsin K complex (Ve of 11 ml) was not affected by dextran.Soluble type I bovine collagen was digested with 800 nM cathepsin K at28° C., 100 mM sodium acetate buffer pH 5.5, containing 0.15% dextranand 2.5 mM EDTA/DTT with/without 0.15% CSA. The reaction was stopped bythe addition of 10 μM E-64 and the samples were analyzed by 4-20%SDS-Page.

[0100] Dextran, a neutral polysaccharide was unable to generatehigh-molecular complexes with cathepsin K and did not interfere with thecomplex formation in the presence CSA. Thus, the collagenolytic activityof the CSA/cathepsin K complex was identical in the presence of dextran.

Example 13 Lack of Complex Formation for Human Cathepsins L, B, and S

[0101] 50 ng of [¹²⁵]DCG-04 labeled cathepsin K Y212C, Cat L, Cat B, andCat S were preincubated with running buffer in the presence or theabsence of 0.1% of CSA, respectively, for 20 min, and then at 37° C.mixed the samples with non-denature protein sample buffer, DNA dye, and37° C. agarose gel (final concentration at 0.4%), and submitted to 0.5%agarose gel in running buffer. The gel was run at about 25° C. 55 V, 250mA for 30 min, then dried and developed the radio signal on a film. Therunning buffer was 125 mM sodium acetate, pH 5.0, 0.5 mM EDTA, 1 mM DTT,40 mM NaCl, and 0.1% chaps. Only cathepsin K formed a stable complex inthe presence of CSA.

Example 14 Lack of Complex Formation with Pycnodysostosis-CausingCathepsin K Mutant, Y212C

[0102] [¹²⁵]DCG-04 labeled wild-type cathepsin K and mutant Y212C wereseparated in the presence or absence of 0.2 μg/ml and 1000 μg/ml CSA,respectively, in a 0.5% agarose gel as described above. Y212C was unableto form a complex with CSA at either low or high CSA concentrations.Type I collagen was incubated with 800 nM of recombinant Y212C and 800nM wild-type cathepsin K in the absence or presence of 0.15% CSA andanalyzed by SDS-polyacrylamide electrophoresis. The result showed thatY212C was not able to hydrolyze type I collagen. Gelatin hydrolysis wasperformed in the presence of 1 nM Y212C, 0.15% CSA at 37 C for 8 h. Theresults showed that Y212C exhibited gelatinase activity.

Example 15 Method for Detection of CSA/Cat K Complex Formation andApplication for High-Through Put Screening (HTS) of Complex FormationInhibitors

[0103] 20 mg cyanogen bromide (CNBr) dissolved in 200 μl deionized waterwere added to 1 ml of 30 mg/ml chondroitin 4-sulfate (CSA). The pH wasadjusted to pH 11 by addition of 5 M NaOH. Activated CSA is applied to aPD-10 column and eluded with 200 mM sodium borate buffer, pH 8.0. CSAfractions were directly dropped into 400 μl of 10 mg/ml fluoresceinaminein 200 mM sodium borate buffer, pH 8.0. The mixture was kept overnightat 4° C. for the coupling reaction. Subsequently, the mixture wasconcentrated to 1 ml using a SpeedVac concentrator and purified on aPD-10 column and eluded by 50 mM sodium acetate buffer, pH 5.5.Fluoresceinamine-labeled CSA (FL-CSA) fractions were collected and theirconcentration was determined using the Blyscan glycosaminoglycan assaykit (Biocolor Ltd., Newtown Abbey, Northern Ireland).

[0104] Fluoresceinamine-labeled CSA/Cat K complex formation was detectedin a Perkin-Elmer fluorimeter at excitation and emission wavelengths of300 nm and 604 nm, respectively. Cat K at a final concentration of 120nM was added to 1 ml 100 mM sodium acetate buffer, pH 5.5 containing 2mM EDTA/DTT using 2 milliliter cuvettes. The fluorescence signal for theprotein alone is close to zero. After the addition offluoresceinamine-labeled CSA at a final concentration of 0.6 μg/ml, thefluorescence signal increased reaching a saturation maximum after 5-10min. This is expected to reflect the generation of the catK/labeled CSAcomplex. Like the protein alone, fluoresceinamine-labeled CSA does nothave a fluorescence signal by itself at the wavelengths used for theassay. After the addition of complex formation inhibitors, thefluorescence signal dropped to zero (FIG. 1). This assay allows thesimple and convenient screening of compounds capable to interfere withcomplex formation. If the fluorescence signal significantly decreasesafter adding a certain concentration of a compound (e.g., 6 μg/ml ofdextran sulfate) this compound can be selected as a potential inhibitorof CatK/CSA complex formation.

[0105] The following test compounds have been demonstrated to decreasethe fluorescence signal: dextran sulfate, dermatan sulfate, heparansulfate, poly-glutamic acid, poly-aspartic acid (results not shown). Incontrast, dextran, poly-lysine, poly-alanine, and chondroitindisaccharide-4S did not show any effect on the fluorescence signal. Allcompounds decreasing the fluorescence signal have been independentlydemonstrated to form a complex with catK as shown by the agarose gelelectrophoretic mobility shift assay and gel filtration and to block orsignificantly decrease the collagenolytic activity of the catK/CSAcomplex. Compounds not affecting the fluorescence signal did not reveala gel mobility shift or a decrease of the collagenolytic activity of theprotease complex.

Example 16 High Throughput Assay for Screening of Complex FormationInhibitors

[0106] The single cuvette assay described above is translated into a 96well format allowing the automation of the screening process. Thefollowing protocol is followed: 1 positive control (100 nM Cat K, 0.5μg/ml FL-CSA, and 5 μg/ml of dextrane sulfate), 1 negative control (100nM Cat K+0.5 μg/ml Fl-CSA), and two concentrations of the test compound(100 nM Cat K+0.5 μg/ml and 10 μg/ml test compound) in separate wellswith 200 μl of 100 mM sodium acetate buffer, pH 5.5 containing 2 mMEDTA/DTT. The samples are mixed using the automixer and the fluorescencesignal is monitored in a microplate fluorescence reader (Gemini XS)using the excitation and emission wavelengths of 300 nm and 604 nm,respectively. If the signal is significantly decreased in a testing wellcomparing to the negative control, the compound can be selected forfurther evaluation of its complex formation capabilities.

Example 17 Identification of Potential Binding Sites for ChondroitinSulfate on Cathepsin K Polypeptide

[0107] Using protein alignment and structure modeling strategies, twopotential sites of glycosaminoglycan and cathepsin K interactions wereidentified and analyzed by site-directed mutagenesis. The followingbasic amino acid residues (lysines (K) and arginines (R)) were found tobe unique for cathepsin K and located in two clusters on the backside ofthe protease (opposing the active site cleft): K77, R79, K103, K106,R108, R111, K122, R127 (cluster 1); R8, K9, K10, (cluster 2). The triplebasic cluster (R8,K9, K10) fits the basic amino acid residue motif knownto bind sulfate glycosaminoglycans.

[0108] The mutant proteins were generated to characterize cluster1:K77A, R79A (mutant 1), K103, K106A, R108A, R111A, (mutant 2), K77A,R79A, K103, K106A, R108A,R111A (mutant 3), K77A, R79A, K103, K106A,R108A,R111A, K122A, R127A (mutant 4). Mutant proteins were expressed inPichia pastoris and the activity of the protein was determined towardstwo synthetic peptide substrates, gelatin and type I native collagen.Wild-type cathepsin K and mutant proteins were incubated for 8 h at 28 Cat a concentration of 800 nM with type I collagen (0.4 mg/ml) in sodiumacetate buffer, pH 5.5 containing 2.5 mM DTT/EDTA in the presence of0.15% CSA. The cleavage products were separated by SDS polyacrylamideelectrophoresis using 4-20% Tris Glycine gels. Wild-type cathepsin K andmutant 1 reveal a complete degradation of type I collagen whereasmutants 2, 3, 4 and 5 are strongly inhibited.

[0109] Mutant proteins and wild type displayed only minor or nodifferences in their activity towards the synthetic peptide substratesand gelatin. In contrast a significant inhibition of theircollagenolytic activity was observed for mutants 2, 3 and 4. The degreeof inhibition for all three mutants was comparable suggesting that theresidues important for the collagenolytic activity are located inresidues K103, K106A, R108A, R111A (mutant 2). Mutant 1 (K77A, R79A) didnot reveal an inhibition, thus excluding both sites. Mutants 3 and 4 arecumulative mutants of mutants 1, 2 and mutant 3 plus residues K122 andR127. Results obtained from gel-mobility assays did not revealprevention of complex formation, however, suggesting that either cluster1 or 2 are individually sufficient for complex formation. However, theutilization of only one of the binding motifs or altemative bindingmotifs appears to be insufficient to maintain the overall structure ofthe collagenolytically active cathepsinK complex with CSA, thuspreventing or significantly reducing its collagenolytic activity. Thesecomplexes are designated as “non-productive complexes” similar to thecollagenolytically inactive complexes of cathepsin K observed in thepresence of dermatan or dextran sulfate.

[0110] A preliminary crystal structure of cathepsin K in the presence ofCSA revealed interactions with the predicted triple basic motif (R8, K9,K10) in cluster 2. The structure did not show interactions with cluster1 and furthermore revealed a ratio of 1:n (n+10 or more depending on thelength of CSA used in the crystallization experiment) between CSA andcathepsin K. This ratio is in contrast to the 1:1 ratio obtained forcatK/CSA complexes in solution which as collagenolytically active. Itwas assumed that the crystal structure is a crystallization artifactwhich nevertheless may reveal partial interactions between the cathepsinK protein and CSA. In particular, residue K9 showed close contacts witha sulfate group of CSA. Replacing this residue with glutamic acid(mutant 5 (K9E; Glu is present in cathepsin L at this position andcathepsin L does not form complexes with CSA) resulted in a proteinwhich was fully active towards synthetic peptide substrates and gelatinbut significantly reduced its activity towards type I collagen (resultsnot shown). Similar to the mutations in cluster 1, the cluster 2 mutantstill revealed complex formation in the presence of CSA in the mobilityshift assay indicating again that disruption of interaction within theindividual clusters is insufficient for a total abolition of complexformation.

[0111] The present invention is not to be limited in terms of theparticular embodiments described in this application, which are intendedas single illustrations of individual aspects of the invention.Functionally equivalent methods and apparatus within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications and variations are intended to fall withinthe scope of the appended claims.

We claim:
 1. A purified collagenolytically active cathepsin K complexcomprising cathepsin K and at least one glycosaminoglycan.
 2. Thepurified cathepsin K complex of claim 1, comprising 5 cathepsin Kmolecules per molecule.
 3. The purified cathepsin K complex of claim 1,wherein the glycosaminoglycan is selected from the group consisting ofchondroitin sulfate keratan sulfate.
 4. A pharmaceutical compositioncomprising the cathepsin K complex of claim
 1. 5. A kit comprising thepharmaceutical composition of claim
 4. 6. A purified antibody whichbinds specifically to the cathepsin K complex of claim
 1. 7. Thepurified antibody of claim 6, wherein the antibody is a monoclonalantibody.
 8. A pharmaceutical composition comprising a therapeuticallyeffective amount of the antibody of claim 6 and a pharmaceuticallyacceptable carrier.
 9. A kit comprising the pharmaceutical compositionof claim
 8. 10. A method of screening for agents that modulate theformation of collagenolytically active cathepsin K complex, comprising:(a) contacting a candidate agent with the a medium containingnon-complexed components of the collagenolytically active cathepsin Kcomplex, and (b) quantitatively detecting formation of acollagenolytically active cathepsin K complex.
 11. The method of claim10, wherein the non-complexed components are monomeric cathepsin K andglycosaminoglycans.
 12. A method of screening for or identifying agentsthat interact with a cathepsin K complex, comprising (a) contacting acandidate agent with a cathepsin K complex, and (b) quantitativelydetecting interaction between the candidate agent and the cathepsin Kcomplex.
 13. The method of claim 12, wherein the step of quantitativelydetecting comprises testing at least one aliquot of the sample, saidstep of testing comprising: (a) contacting the aliquot with an antibodythat is immunospecific for a cathepsin K complex, and (b) quantitativelymeasuring any binding that has occurred between the antibody and atleast one species in the aliquot.
 14. The method of claim 12, whereinthe interaction is binding.
 15. A method of screening for or identifyingagents that modulate the activity of a collagenolytically activecathepsin K complex, comprising (a) in a first aliquot, contacting acandidate agent with the collagenolytically active cathepsin K complex,and (b) comparing the activity of the collagenolytically activecathepsin K complex in the first aliquot after addition of the candidateagent with the activity of the collagenolytically active cathepsin Kcomplex in a control aliquot, or with a previously determined referencerange.
 16. The method of claim 15, wherein activity of thecollagenolytically active cathepsin K is measured in a test sample in amethod comprising the steps of: a) incubating said test sample with asubstrate for collagenolytically active cathepsin K complex for apredetermined period of time, and b) determining the amount of substratepresent after the predetermined period of time.
 17. The method of claim15, wherein modulation is inhibition of collagenolytic activity.
 18. Themethod of claim 15, wherein modulation is enhancement of collagenolyticactivity.
 19. A method of screening for or identifying agents thatmodulate the formation or activity of a cathepsin K complex, comprising:(a) administering a candidate agent to a first mammal or group ofmammals; (b) administering a control agent to a second mammal or groupof mammals; and (c) comparing the amount or activity of the cathepsin Kcomplex in the first and second groups, or comparing cellular responseto cathepsin K complex in the first and second groups.
 20. A method oftreating or ameliorating a cathepsin K complex-mediated disorder,comprising administering to the patient a therapeutically effectiveamount of a reagent that modulates cathepsin K complex activity.
 21. Themethod of claim 20, wherein the cathepsin K complex-mediated disorder isselected from the group consisting of osteoporosis, skin aging, Paget'sdisease, and rheumatoid arthritis.
 22. The method of claim 20, whereinthe cathepsin K complex-mediated disorder results from excessivedegradation of extracellular bone and/or cartilage matrix.
 23. Themethod of claim 20, wherein the cathepsin K complex-mediated disorderresults from insufficient degradation of extracellular bone and/orcartilage matrix.